The overall goal of this post-doctoral fellowship is to elucidate the signaling mechanisms by which dopamine (DA) D2 receptor (D2R) activation modulates dopamine transporter (DAT) function and expression. The proposed experiments will test the hypotheses that (1) the biochemical mechanisms underlying the D2R mediated increase in DAT function and cell surface expression is through G-beta gamma-subunit interactions with the mitogen activated protein kinase (MAPK) pathway, and (2) the effects of D2R stimulation on DAT in postnatal DA neuronal cultures will be similar to those observed in oocytes. D2R activation causes a rapid increase in the velocity of DA uptake and the number of cell surface DATs in Xenopus oocytes co-expressing human (h) D2Rs and DATs. First, we will determine the role of the G-beta gamma subunit in the D2R-mediated effects on hDAT function ([3H]DA uptake, hDAT-associated currents) and expression ([3H]WIN 35428 binding) in oocytes. Next, we will examine the involvement of the MAPK pathway in these D2R-mediated effects on hDAT function and expression in oocytes. Finally, we will characterize the effects of D2R stimulation on DAT function in postnatal DA neuronal cultures. Once established, this neuronal culture system will be very valuable for further studies elucidating the mechanisms by which drugs of abuse alter DAT regulation. These experiments will enhance our understanding of the signaling mechanisms mediating D2R regulation of DAT, the target of important drugs of abuse and a critical component responsible for modulating dopaminergic synaptic neurotransmission.